CN104258397A - Function and application of Dickkopf-3 for treating atherosclerosis - Google Patents

Abstract

The invention discloses a function and application of Dickkopf-3 (DKK3) for treating atherosclerosis and belongs to the fields of gene function and application. inducing by taking ApoE-/-mice and DKK3-/-ApoE-/-mice taken as experimental subjects are induced high-fat diet to obtain an atherosclerosis model, and results show that compared with the ApoE-/-mice, DKK3 gene defect significantly reduces plaque area of aorta, enhances stability of aortic sinus plaque and remarkably relieves an inflammatory reaction, indicating that DKK3 in atherosclerosis mainly takes a role in promoting formation of aortic plaque, in particular in promoting atherosclerosis. For the function, DKK3 can be used as a drug target for screening medicines for preventing, relieving and/or treating atherosclerosis; and a DKK3 inhibitor can be used for preparing a medicine for preventing, relieving and/or treating atherosclerosis.

Description

The function and application of Dickkopf-3 in treatment atherosclerosis

Technical field

The invention belongs to function and the application of gene, be specifically related to a kind of Dickkopf-3(DKK3) treating the function and application in atherosclerosis, specifically DKK3 prevents in preparation, alleviates and/or treat in atherosclerotic medicine and apply. background technology

Cardiovascular and cerebrovascular disease be main lethal in many developed countries because of, also raise year by year at the sickness rate of China and fatality rate.The basis of cardiovascular and cerebrovascular disease is atherosclerosis (Atheosclersisis, AS), and atherosclerosis can make ductus arteriosus wall thicken, hardening, luminal stenosis, causes a lot of cardiocerebrovasculaevents events to occur.And the coronary artery acute stenosis that causes of the breaking of atherosclerosis unstable spot, hematoblastic gathering and thrombosis and obturation are the major reasons causing acute coronary syndrome (acute coronary syndrome, ACS).

Atherosclerosis is multiple gene, a kind of chronic inflammation disease that risk factor and immunologic mechanism participate in jointly, the inflammatory and immune response of local and whole body plays an important role in atherosclerosis generation evolution, inherent immunity and adaptive immunity participate in regulating atherosclerotic lesion jointly, pathology show as large, medium-sized artery many places Mottling formation, be apt to occur in blood shunt, the regions such as the bending and arterial branch of tremulous pulse, its characteristics of lesion is that in blood, lipid deposits at endarterium, cause inner membrance stove fibrous thickening, focus deep is the medicated porridge sample material formed by slough and extracellular lipid pond.Panimmunity cell is there is in atheromatous plaque, wherein with macrophage and T cell the most common, in addition a small amount of dendritic cell (Dentritic cell is also had, DC), natural killer cell (natural killer cell, NK cell) and mastocyte (mast cell) etc., occasionally there is bone-marrow-derived lymphocyte.

The macroscopic damage of atherosclerosis earliest period is fatty streaks, forms primarily of the Macrophage derived foamy cell that intake of a large amount of cholesterol.Mononuclear cell in blood circulation is attached to reactive endothelial cells at tremulous pulse damageable zone, starts the formation of fatty streaks, under the attraction that the mononuclear cell sticked is subject to the chemistry of local generation to ingratiate with molecule subsequently moves to inner membrance, and is divided into macrophage further.A large amount of cholesterol ester, in macrophage inner accumulated, forms foam cell, and this is the early stage characteristic pathological physiological process of atherogenesis.Atherosclerotic is multifactor coefficient result, and the atherosclerosis risk sexual factor found at present is a lot, but related pins to treatment and control effects all undesirable, blood fat reducing and anti-inflammatory treatment are current topmost remedy measures.

Wnt albumen is one group of glycosylated protein being rich in cysteine, participates in the propagation of cell, differentiation, apoptosis and controls the process such as location of cell.Pericyte (pericytes) is main endothelium sustenticular cell, and near EC, it has the maturation of regulation and control EC, stablizes wall of micrangium and promote the functions such as new vessels and blood vessel sprout.Research shows to fatty series differentiation, Wnt signal suppressing pericyte promotes that it is to the differentiation of cartilage system, and prompting Wnt signal path may relevant with the generation of AS [1].There is a large amount of Wnt5a in the region issuing the accumulation of present macrophage in apo E (ApoE) deficient mice and the carotid artery intima of the patient accepting endarterectomy, shows that Wnt5a has expression [2] in the mankind and mice AS pathological changes.DKK3 is one of DKK family member.DKK family is by 5 member compositions: DKK1, DKK2, DKK3, DKK4 and DKK3 associated protein soggy(sgy), be the antagonizes factor of Wnt signal pathway, carried out the intracellular signaling in regulating cell by its corresponding receptors bind.DKK1-4 is all rich in cysteine regions containing two, and 10 cysteine residues containing high conservative between family member in region, this region is the significant structure [3] of DKK albumen.Dickkopf-3(DKK3) be a kind of secretory protein of Dickkopf family, there are some researches show that DKK3 plays an important role at the growth of organization of regulation control, apoptosis, propagation and immunology.DKK3 can wide expression at different tissues, comprise heart tissue, be proved in heart tissue, DKK3 is by regulating ASK1-JNK/p38 signal path, thus protection pathological cardiac hypertrophy [4].Based on above-mentioned Research foundation, we think that DKK3 may play key player in the characteristic pathological physiological process of atherogenesis, but its biological function in the characteristic pathological physiological process of atherogenesis and possible mechanism of action are not yet illustrated.

Mice and genetic engineering mice resource are one of most important animal models in field such as current disease mechanisms research, gene functional research, medicine initiative, are also the necessary conditions of these field innovation researches.Utilize genetic engineering mice to study for each molecule link in Atheromatosis reason process, to the molecular mechanism illustrated in atherosclerosis generation evolution, the molecular target finding treatment of atherosclerosis has great importance.

[list of references]

1、Kirton JP,Crofts NJ，George SJ,et al.Wnt/ beta catenin signaling stimulates chondrogenic and inhibits adipogenic differentiation of pericytes：potential relevance to vascular disease Circ Res, 2007; 101(6): 581-9.

2、Christman MA 2nd，Goetz DJ，Dickerson E,et al.Wnt5a is expressed in murine and human atherosclerotic lesions. Am J Physiol Heart Circ Physiol, 2008; 294(6): 2864-70.

3、Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene. 2006; 25(57): 7469-81.

4、Zhang Y, Liu Y, Zhu XH, et al. Dickkopf-3 attenuates pressure overload-induced cardiac remodelling. Cardiovasc Res. 2014; 102(1): 35-45。

Summary of the invention

The object of the invention is to determine the expression of DKK3 and atherosclerotic mutual relation, provide one for preventing, alleviating and/or treat the novelty teabag of atherosclerotic target gene DKK3.

Object of the present invention is achieved through the following technical solutions:

Expression and the atherosclerotic relation of the DKK3 that the present invention determines are as follows:

1. DKK3 gene knockout significantly reduces atherosclerotic plaque area

The present invention is with ApoE gene knockout (ApoE ^-/-) mice and DKK3/ApoE is dual-gene knocks out (DKK3 ^-/-apoE ^-/-) mice is experimental subject, obtains atherosclerosis mouse model (AS) respectively by low fat feedstuff and high lipid food diet induced.Result shows ApoE ^-/-and DKK3 ^-/-apoE ^-/-mice is by low fat feed diet, and aorta tree has a small amount of Mottling formation; After high lipid food diet, plaque area significantly increases, DKK3 ^-/-apoE ^-/-the aorta tree of mice, the plaque area of aortic sinus are all significantly less than ApoE ^-/-mice (Fig. 1-2).

2. DKK3 gene knockout significantly strengthens the stability of aortic sinus speckle

The present invention is with ApoE gene knockout (ApoE ^-/-) mice and DKK3/ApoE is dual-gene knocks out (DKK3 ^-/-apoE ^-/-) mice is experimental subject, obtain atherosclerosis mouse model (AS) by high lipid food diet induced, the stability of aortic sinus speckle is studied.Result shows that DKK3 gene knockout significantly reduces the content of macrophage and the area of necrotic center in aortic sinus speckle, increases content and the collagen component content of smooth muscle cell in speckle, enhances the stability (Fig. 3) of aortic sinus speckle.

3. DKK3 gene knockout significantly reduces inflammatory reaction

The present invention is with ApoE gene knockout (ApoE ^-/-) mice and DKK3/ApoE is dual-gene knocks out (DKK3 ^-/-apoE ^-/-) mice is experimental subject, obtain atherosclerosis mouse model (AS) by high lipid food diet induced, the expression of the inflammatory factor of aortic sinus speckle is studied.Result shows that DKK3 gene knockout significantly reduces the proinflammatory factors such as ICAM-1 and IL-6 of aortic sinus speckle, increases the expression (Fig. 4) that IL-10 etc. presses down the scorching factor.

During generation atherosclerosis known by above result, DKK3 genetic flaw decreases plaque area, strengthens the stability of aortic sinus speckle, significantly reduces inflammatory reaction.Therefore DKK3 has the effect promoting atherosclerosis generation development, for the research atherosclerotic novel targets of control and New Policy provide theoretical foundation and Clinical Basis.

Therefore, DKK3 gene can be used as drug target, builds In vitro cell model or the animal model of DKK3 gene overexpression, for screening prevention, alleviating and/or treating atherosclerotic medicine; DKK3 gene also can be used as the target gene in gene therapy, designs and prepares prevention, alleviates and/or treat atherosclerotic medicine and/or biological reagent, reaching prevention, alleviate and/or treat atherosclerotic object by technique for gene engineering.Be such as target gene with DKK3, design the double-strand siRNA that DKK3 can be disturbed to express, after being synthesized by chemical method, be injected into human body and make DKK3 gene silencing treat atherosclerosis by the method that RNA disturbs; Can also design and build the mutant of DKK3, after injection, entering cell, the substrate specificity of competition DKK3 original shape, thus suppressing the function of DKK3, playing therapeutic purposes; In addition, can also be shot design micromolecular compound inhibitor with DKK3, utilize In vitro cell model or the animal model of DKK3 gene overexpression, by screening, find wherein to suppress the molecule of DKK3 by specificity, thus provide new therapeutic molecules for atherosclerotic treatment.

For the above-mentioned functions of DKK3, DKK3 is provided to prevent as drug targets in screening, alleviate and/or treat the application in atherosclerotic medicine.

For the above-mentioned functions of DKK3, provide the inhibitor of DKK3 in preparation prevention, alleviate and/or treat the application in atherosclerotic medicine.

One is prevented, is alleviated and/or treat atherosclerotic medicine, comprises the inhibitor of DKK3.

The inhibitor of described DKK3 is preferably the rna interference vector of siRNA, DKK3 gene of DKK3 gene, the one in the antibody of DKK3 and other inhibitor that DKK3 can be suppressed to express.

The present invention has following advantage and effect relative to prior art:

1. the present invention finds the New function of DKK3 gene, and namely DKK3 can promote atherosclerotic effect.

2. promoting atherosclerotic function based on DKK3, providing target for developing atherosclerotic medicine.

3. the inhibitor of DKK3 can be used for preparation prevention, alleviates and/or treat atherosclerotic medicine.

Accompanying drawing explanation

Fig. 1 is ApoE ^-/-and DKK3 ^-/-apoE ^-/-the aorta tree oil red O stain of mice and plaque area cartogram, result shows that DKK3 gene knockout significantly reduces the plaque area (NS: there was no significant difference) of aorta tree.

Fig. 2 is ApoE ^-/-and DKK3 ^-/-apoE ^-/-mouse aorta hole HE dyes and plaque area statistics block diagram, and result shows that DKK3 gene knockout significantly reduces plaque area (*: the p < 0.05 vs HFD ApoE of aortic sinus ^-/-group).

Fig. 3 is ApoE ^-/-and DKK3 ^-/-apoE ^-/-the dyeing of necrotic center, collagen component, macrophage and smooth muscle cell content and result statistics block diagram in the aortic sinus speckle of mice, result shows that DKK3 gene knockout significantly reduces the content of macrophage and the area of necrotic center in aortic sinus speckle, increases content and collagen component content (*: the p < 0.05 vs HFD ApoE of smooth muscle cell in speckle ^-/-group).

Fig. 4 is ApoE ^-/-and DKK3 ^/-apoE ^-/-the expression immunofluorescence dyeing of the inflammatory factor such as ICAM-1, IL-6, IL-10 and result statistics block diagram in the aortic sinus speckle of mice, result shows that DKK3 gene knockout significantly reduces the expression of proinflammatory factor ICAM-1 and IL-6 of aortic sinus speckle, increases expression (*: the p < 0.05 vs HFD ApoE of anti-inflammatory factors IL-10 ^-/-group).

Detailed description of the invention

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

Animal for research and raising

Laboratory animal kind, sex, age in week and source: ApoE gene knockout (ApoE ^-/-) mice and DKK3/ApoE is dual-gene knocks out (DKK3 ^-/-apoE ^-/-) mice, male, 8 week age, body weight 19-25g.ApoE knock out mice (ApoE ^-/-, purchased from Jackson Laboratory, article No. 002052); DKK3 ^-/-apoE ^-/-mice is obtained by DKK3 knock out mice (purchased from Japanese RIKEN company, article No.: RBRC02847) and the hybridization of ApoE knock out mice.

Laboratory animal feed formula: high lipid food (HFD, purchased from Fukang bio tech ltd of China, Beijing, by AIN-76 A Western Diets formula, percent of calories: protein 15.8%, fat 40%, carbohydrate 44.2%); Low fat feedstuff (NC, purchased from Fukang bio tech ltd of China, Beijing, article No. D12450B): percent of calories: protein 20%, carbohydrate 70%, fat 10%.

Animal feeding and environmental condition: all experiment mices are all raised at angiocardiopathy institute of Wuhan University SPF level Animal House (credit number: SYXK(Hubei Province): 2009-0053).Alternately illumination in every 12 hours, temperature 24 ± 2 DEG C, humidity 40%-70%, mice freely drinks water feed.

Embodiment 1 rat aorta is atherosis, and model (AS) obtains

1. laboratory animal grouping: select 8 week age, body weight 19-25g, male, ApoE ^-/-mice and DKK3 ^-/-apoE ^-/-mice, gives high lipid food (Western Diets, HFD) respectively and low fat feedstuff (Normal chow, NC) is raised, ApoE ^-/-hFD group, ApoE ^-/-nC group, DKK3 ^-/-apoE ^-/-hFD group, DKK3 ^-/-apoE ^-/-nC group is totally 4 groups, often organizes each 20.

2. Atherosclerosis Model induces operating process by high lipid food:

Adopt ApoE ^-/-mice and DKK3 ^-/-apoE ^-/-mice, sets up AS model, carries out phenotype correlation analysis, specifies the effect that DKK3 gene pairs atheromatosis plays.Mice was from 8 week age, and the omnidistance high lipid food of HFD group is fed and put to death and collect sample for 28 weeks, and the omnidistance low fat forage feed of NC group is put to death and collects sample for 28 weeks.

Embodiment 2 AS model mice plaque area measures

1. mice last tissue sampling eventually

Mice feed high fat or low fat feedstuff until 28 weeks time, weigh, use 3% pentobarbital sodium, 90mg/kg anesthetized mice, be fixed on material drawing board with syringe needle, with the moistening mice skin of chest abdomen of gauze, cut off thoracic cavity with eye scissors, expose heart, cut off right auricle, the syringe needle of transfusion device is lunged left ventricle, slowly inject 10-15mL PBS buffer with 50mL syringe, treat that right auricle effluent is limpid, change 4% paraformaldehyde and continue to inject 10-15mL.After perfusion terminates, remove splanchnocoel internal organs, only retain heart.Under mice is placed in microscope, is separated fascia, fatty tissue around aortic arch, cut heart in ascending aorta initial part, cut off in the middle part of thoracic aorta, and about 3mm place cuts off under neck summation clavicle, aortic arch is put into above-mentioned EP pipe.

2. aorta tree plaque area measures

Aorta tree be placed in 4% paraformaldehyde overnight fixing → pure water rinsing 30min → 60% isopropyl alcohol process 10 min → oil red O dye liquor (company sigma, article No. 00625) dyeing 60min → 60% isopropyl alcohol 1min × 3 time remove remaining outer wall fat → to be laid in by tremulous pulse dye on black dissection stencil plate to clean background → anatomical lens, take pictures with digital camera after dyeing, and use Image-Pro Plus 6.0 image analysis software to carry out plaque area quantitative assay (oil red O stock solution=0.5 gram oil red O+100 milliliter 100% isopropyl alcohol, oil red O dye liquor (working solution): V(oil red O stock solution)/V(H ₂o)=3/2).

Aorta tree plaque area (%)=speckle gross area/aorta tree gross area * 100%.

3. pathological tissue process

3.1 paraffin specimen preparations

In 4% paraformaldehyde overnight fixing after, aortic arch filter paper is carefully wrapped, in case spill from embedding frame gap.Dewaterer is put into, 30% ethanol 15min → 50% ethanol 15min → 75% ethanol 15min → 85% ethanol 15min → 95% ethanol 15min → 100% ethanol 15min → 100% ethanol 15min → dimethylbenzene 15min → dimethylbenzene 15min → paraffin 30min → paraffin 30min after running water 30min.After aortic arch has dewatered, take out from dewaterer, aortic arch lies low in paraffin.

3.2 aortic tissue sections

Use microtome to aortic sinus paraffin specimen section (slice thickness 5 μm).

4. aortic sinus plaque area measures

Haematoxylin eosin stains (HE dyeing): get the moisture of aortic sinus paraffin white tiles 65 DEG C baking 30 minutes → dimethylbenzene 5 minutes × 3 times → 100% ethanol 1 minute → 95% ethanol 1 minute → 70% ethanol, 1 minute → pure water rinsing (slide not to hang with the globule for standard) → get rid of on most microscope slide, haematoxylin solution (Zhuhai shellfish rope, BA-4021) contaminate 5 minutes → tap water rinse (remove slide on haematoxylin loose colour) → 1% acidic alcohol 1-3 second → tap water rinse (removing acidic alcohol on slide) → Scott liquid (sodium bicarbonate 0.35g, magnesium sulfate 2g, distilled water 100mL) 1 minute → tap water embathes (removing the Scott liquid on slide) → get rid of the moisture be all on slide, Yihong solution (Zhuhai shellfish rope, BA-4024) → pure water rinsing (removing the loose colour in Yihong on slide) → 70% ethanol once → 95% ethanol once → 100% ethanol that dyes for 10 seconds 30 seconds, 3 times → dimethylbenzene 2 minutes, 3 times → take advantage of dimethylbenzene not dry time mounting (with bubble-free of cutting into slices for principle) → fume hood in dry up, microscope is taken pictures.

HE dyeing picture statistics: directly by Image-Pro Plus 6.0 image analysis software circle aortic sinus plaque area.

By aorta tree oil red O stain substantially can assess that atheromatous plaque on whole piece blood vessel formed number, distribution situation and plaque area size.Fig. 1 is mice AS model aorta posterior tree oil red O stain result figure, and brachiocephalic trunk and aortic sinus are the most obvious positions of atheromatous plaque, ApoE ^-/-and DKK3 ^-/-apoE ^-/-mice is by low fat feed diet, and aorta tree just has a small amount of Mottling formation, and DKK3 ^-/-apoE ^-/-the plaque area of mice and ApoE ^-/-mice is without significant difference; After high fat diet, plaque burden significantly increases, DKK3 ^-/-apoE ^-/-the aorta tree plaque area of mice is significantly less than ApoE ^-/-mice.Fig. 2 is mice AS model posterior sinus of Valsalva HE coloration result figure, and result shows DKK3 after high fat diet ^-/-apoE ^-/-the plaque area of the aortic sinus of mice is significantly less than ApoE ^-/-mice.Above result shows that DKK3 gene knockout significantly reduces atherosclerotic plaque area.

The mensuration of embodiment 3 AS model mice plaque stability

1. the size of aortic sinus necrotic center measures

Haematoxylin eosin stains (HE dyeing), method is with embodiment 2.4, and the microscope of organizing chosen containing cholesterol crystal, acellular core fibre structure is taken pictures.

The area estimation of necrotic center: use Image-Pro Plus 6.0 image analysis software circle necrotic center area.

2. aortic sinus collagen component assay:

Picro-Sirius red (PSR) dyes, key step is: get aortic sinus paraffin white tiles 55 DEG C baking 30min → dimethylbenzene 2min, 3 times → 100% ethanol 1min → 95% ethanol 1min → 70% ethanol 1min → running water 10min → distilled water 1min → mass fraction 0.2% phosphomolybdic acid 2min → 0.1% sirius red picric acid solution drips in tissue, dye in wet box 90min → removal residual liquid → 0.01N hydrochloric acid 4s → 70% ethanol 1 time → 90% ethanol 1 time → 100% ethanol 30s, 3 times → dimethylbenzene 2min, 3 times → take advantage of dimethylbenzene not dry coverslip immediately mounting, microscope is taken pictures.

Collagen ratio measuring: use the red area of collagen of Image-Pro Plus 6.0 image analysis software circle, collagen ratio (%)=area of collagen/plaque area * 100%.

3. the expression of macrophage and smooth muscle cell mark measures

Immunofluorescence dyeing detects aortic sinus macrophage marker CD68, smooth muscle cell mark SMA(Smooth Muscle Actin) expression.Required primary antibodie information: CD68(MCA1957; 1:100; Rat; AbD Serotec), SMA(ab5694; 1:100; Rabbit; Abcam); Required two anti-information: Alexa Flour 568 goat anti-rat IgG(A11077; Invitrogen), Alexa Fluor 488-conjugated goat anti-rabbit IgG(A11008; Invitrogen).

Key step is:

1) roasting sheet: aortic sinus paraffin white tiles is placed in more than baking box 30min.

2) dewax: dimethylbenzene 5min × 3 time.

3) hydration: 100% ethanol 5min × 2 time; 95% ethanol 5min; 70% ethanol 5min; ddH ₂o embathes 5min × 2 time.

4) citrate tissue antigen recovery (Pressure method): get a certain amount of pH6.0 citrate antigen retrieval working solution in reparation box, put into pressure cooker, big fire is heated to boiling, tissue slice after dewaxing hydration is placed in and repairs box, cover pot cover, buckle pressure valve, continue to be heated to jet, after starting timing 5min, take out and repair box; Room temperature places 20min, takes out section after natural cooling.

5) ddH ₂o rinsing 5min × 2 time, PBS rinsing 5min × 2 time.

6) groupization stroke circle, drips 10% sheep blood serum (GTX27481, GeneTex) and closes, 37 DEG C of closed 60min in wet box.

7) abandon confining liquid, drip the primary antibodie of proper proportion dilution, 4 DEG C of overnight incubation, 37 DEG C of rewarming 30min, discard primary antibodie, PBS washes 10min × 3 time.

8) drip two to resist, in wet box, hatch 60min for 37 DEG C, discard two and resist, PBS embathes 5min × 3 time.

9) SlowFade Gold antifade reagent with DAPI(S36939, Invitrogen) mounting.

10) viewed under fluoroscopy, takes pictures.

Fluorescent quantitation is added up: use Image-Pro Plus 6.0 image analysis software to measure Positive Cell Counts, CD68/SMA(%)=positive cell number/plaque area * 100%.

Macrophage is most important cell component in speckle, it mainly contain blood circulation mononuclear cell enter interior subcutaneous after differentiate, Monocytes/Macrophages can secrete multiple adhesion, chemotactic factor as cell adhesion molecule (ICAM-1), MCAF (MCP-1) etc., promote entering of speckle inner cell, in addition macrophage also can secrete multiple matrix metalloproteinase (MMPs), reduce area of collagen in speckle, thus destroy the stability of speckle; Smooth muscle cell can secrete various kinds of cell substrate, is the cell source of atheromatous plaque endocrine collagen, and Main Function repairs destroyed cellular stromal component thus plays a protective role; Collagen component is most important extracellular matrix in speckle, is also the main component of fibrous cap, and having anti-blood flow and impact the effect preventing plaque rupture, is also the important evaluation index safeguarding plaque stability.

Fig. 3 is ApoE ^-/-mice and DKK3 ^-/-apoE ^-/-the analysis result figure of the aortic sinus speckle inclusions of mice.Aortic sinus HE dyes visible DKK3 ^-/-apoE ^-/-the necrotic center area of mice is significantly less than ApoE ^-/-mice; PSR coloration result shows the model after high fat diet, DKK3 ^-/-apoE ^-/-the collagen ratio of group mice is apparently higher than ApoE ^-/-mice; Immunofluorescence is sent out and is observed macrophage marker CD68 at DKK3 ^-/-apoE ^-/-expression in mice speckle is then remarkable in ApoE ^-/-mice; Smooth muscle cell mark SMA is at DKK3 ^-/-apoE ^-/-expression in mice speckle is apparently higher than ApoE ^-/-mice.Above result shows that DKK3 gene knockout significantly reduces the content of macrophage and the area of necrotic center in aortic sinus speckle, increases content and the collagen component content of smooth muscle cell in speckle; Therefore DKK3 gene knockout can strengthen the stability (Fig. 3) of aortic sinus speckle.

The mensuration of inflammatory Cytokines Expression in embodiment 4 AS model mice speckle

Immunofluorescence dyeing detects the expression of the inflammatory factors such as aortic sinus ICAM-1, IL-6, IL-10.Required primary antibodie information: ICAM-1(AF796; 1:100; Goat; R & D systems), IL-6(AF-406-NA; 1:100; Goat; R & D systems), IL-10(AF-217-NA; 1:100; Goat; R & D systems); Required two anti-information: Alexa Flour 568 donkey anti-goat IgG(A11057; Invitrogen).

Key step is: see embodiment 3.3.

Fluorescent quantitation is added up: use Image-Pro Plus 6.0 image analysis software to carry out absorbance (IOD) to positive cell and measure.

Inflammatory reaction is one of principal element causing atheromatous plaque to break.The a large amount of cytokine of proinflammatory cytokine secretion is as interleukin-6 (IL-6), and interleukin 10 (IL-10) etc. can cause the progress of the activation of endotheliocyte, the hypertrophy apoptosis of smooth muscle cell and medicated porridge sample pathological changes.Adhesion, the chemotactic factor of impaired endotheliocyte and Monocytes/Macrophages secretion are the key factors that transmitting inflammation cell is assembled to atheromatous plaque.The expression change of the inflammatory factors such as ICAM-1, IL-6, IL-10 is observed by immunofluorescence dyeing, result shows that DKK3 gene knockout significantly reduces the expression of proinflammatory factor ICAM-1, IL-6 in aortic sinus speckle, increases the expression (Fig. 4) of IL-10 anti-inflammatory cytokine.

Above-described embodiment result shows, ApoE ^-/-mice and DKK3 ^-/-apoE ^-/-mice issues lively pulse atherosclerosis in the induction of high fat diet, and ApoE ^-/-mice is compared, and the dual-gene Aortic Plaque area knocking out rear mice of DKK3/ApoE significantly reduces, and plaque stability also increases, and inflammatory reaction obviously alleviates.These results show, DKK3 significantly can promote the formation of Aortic Plaque and atheroscleroticly to develop.Present invention demonstrates that DKK3 has important deterioration effect in Atherosclerosis Model, its inhibitor can be used for the medicine preparing treatment atheromatosis.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (4)

1.DKK3 prevents as drug targets in screening, alleviates and/or treat the application in atherosclerotic medicine.

The inhibitor of 2.DKK3 prevents in preparation, alleviates and/or treat the application in atherosclerotic medicine.

3. prevent, alleviate and/or treat an atherosclerotic medicine, it is characterized in that: the inhibitor comprising DKK3.

4. application according to claim 2 or medicine according to claim 3, is characterized in that: the inhibitor of described DKK3 is the one in the rna interference vector of siRNA, DKK3 gene of DKK3 gene or the antibody of DKK3 and other inhibitor that DKK3 can be suppressed to express.